Autoantibodies to p53 in ovarian cancer patients and healthy women: a comparison between whole p53 protein and 18-mer peptides for screening purposes.

Autoantibodies against complete p53 protein and 18-mer peptides of p53 in ovarian cancer patients and healthy women were examined. Sera from 9% (4/46) of ovarian cancer patients but none (0/51) of healthy women recognized complete p53 protein. The antibodies were mainly of the IgG1 isotype. Two patients had also IgG2 antibodies. Sera from 28% (13/46) of cancer patients and 21% (11/52) of healthy women contained either IgM, or IgM plus IgG2 antibodies against 18-mer p53 peptides. Screening against complete p53 protein instead of peptides seems necessary for identifying patients with tumor-related antibodies. IgG2 antibodies against p53 suggest p53-specific CD4+ T helper 1 cell activity in some of the ovarian cancer patients.

ME491 melanoma-associated glycoprotein family: antigenic identity of ME491, NKI/C-3, neuroglandular antigen (NGA), and CD63 proteins.

BACKGROUND: Numerous monoclonal antibodies (MAbs) have been produced to antigens found in human melanomas. Three of the best characterized melanoma antigens include the melanoma-associated glycoproteins (MAGs) defined by two reagent families--the ME491 family (including ME491, 8-1H, and 8-2A) and the NKI/C-3 family (including NKI/C-3 and NKI/black-13)--as well as the neuroglandular antigen (NGA) defined by MAbs LS59, LS62, and LS140. These three antigens have significant similarities in tissue distribution, biosynthesis, and structure. The ME491 MAG has been cloned, mapped, and sequenced. Numerous non-melanoma-associated proteins (Sm23, CO-029, R2, TAPA-1, CD9, CD37, CD53, and CD63) have recently been shown to have significant homology to this sequence. PURPOSE: We conducted this study to investigate the similarity between the two MAG antigens and NGA. METHODS: Several reagents defining the three different melanoma antigens were compared, using competition immunoprecipitation, immunoassay, and inhibition radioimmunoassay techniques. RESULTS: Immunoassay experiments show that MAbs defining the three melanoma antigens bind to affinity-purified ME491 antigen and inhibit each other from binding in an inhibition radioimmunoassay. Competition immunoprecipitation experiments demonstrate that the ME491 and NKI/C-3 antibodies bind to NGA. Rabbit anti-ME491 idiotype serum recognizes determinants shared by NKI/C-3 and the anti-NGA MAbs. A competition immunoprecipitation experiment also confirms the identity of CD63, as defined by MAb RUU-SP 2.28, with the three melanoma antigens. CONCLUSION: These data indicate that the MAGs defined by ME491 and NKI/C-3 as well as the anti-NGA antibodies are epitopes of the same molecule, which is identical to CD63 by both immunochemical and molecular genetic investigations. IMPLICATIONS: Our results indicate that the data obtained in studies of these three melanoma antigens may be pooled, and we propose that the molecule recognized by these reagents be classified as CD63.

Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.

Structural heterogeneity of a human melanoma-associated antigen.

The biosynthesis, structure, and topology of a melanoma-associated antigen, previously defined with the monoclonal antibody NKI/C-3 was studied. A polyclonal rabbit antiserum was raised against the antigen with a broader reactivity than the previously used monoclonal antibody NKI/C-3. The antigen was shown to consist of a single protein backbone to which two or three N-linked glycans were added cotranslationally. Extensive further heterogeneity was generated in the Golgi compartment and was shown to be dependent on the presence of complex type sugars. Although the antigen is associated with melanomas, it was not codistributed with the tyrosinase activity associated with melanogenesis. The antigen did show codistribution with cathepsin D, which is a marker for lysosomal functions.

Monoclonal antibodies in detection of choroidal melanoma.

Three monoclonal antibodies (MoAbs) prepared against cutaneous melanomas were tested against one group of 12 choroidal melanomas with indirect immunofluorescence in frozen sections. A fourth MoAb was tested in paraffin sections of a second group of 47 choroidal melanomas. One MoAb (NKI-M7) did not react with choroidal melanoma, even though it had a high sensitivity for cutaneous melanoma. A second MoAb (NKI-M6) showed a positive reaction with only 2/12 choroidal melanomas. The third MoAb (NKI/beteb) reacted with all choroidal melanomas, regardless of the cell type. MoAb NKI/C-3 was positive with 38/47 (81%) choroidal melanomas. We conclude that NKI/C-3 and NKI/beteb have a high sensitivity for both cutaneous and choroidal melanomas in frozen sections. Of these two antibodies NKI/beteb was the most specific for cutaneous naevi and melanomas.

A monoclonal antibody specific for cells of the melanocyte lineage.

A monoclonal antibody, NKI/beteb, was prepared against membranes from a human melanoma metastasis, and in immunoprecipitates of melanoma cell lysates specific 100- and 7-kd glycoproteins were found. The large glycoproteins were also present in conditioned medium of melanoma cell lines. The antigen is located on the inner side of membranes of (pre)melanosomes and premelanosomelike vesicles. The antibody reacted in the immunoperoxidase test on frozen tissue sections with 27 of 28 nevocellular nevi (15/16 common, 12/12 dysplastic), 39/39 primary melanomas (3 intraepidermal, 24 cutaneous, 12 choroidal), 56/63 melanoma metastases, and 4/4 clear-cell sarcomas (melanoma of soft tissue). With sections of formalin-fixed paraffin-embedded tissues, the reaction was less sensitive. No reactivity was detected with frozen sections of 185 other tumors, except for 1 case of non-Hodgkin's lymphoma in which macrophages were positive. With the exception of melanocytes, all frozen sections of adult tissues that were tested were negative with NKI/beteb. On the basis of its tissue distribution so far, the antigen recognized by NKI/beteb seems to be a specific and sensitive diagnostic marker for cells of the melanocyte lineage.

Heterogeneous expression of melanoma-associated antigens in uveal melanomas.

Twenty-five uveal melanomas were stained with seven monoclonal antibodies (MoAbs) recognizing different antigens on cutaneous melanomas. A two step immuno-peroxidase procedure was used. Phenotypic heterogeneity was observed for four MoAbs (M.2.2.4, AMF-7, 225.28S, PAL-M1) while two MoAbs (NKI-beteb, CL203.4) reacted strongly with most (85%) uveal melanomas, and one MoAb (R-24) showed very low reactivity. Despite heterogeneity, co-expression of some antigens was observed. Expression of the antigen recognized by MoAb M.2.2.4. was significantly lowered by pre-enucleation irradiation while the other antigens remained unchanged. Expression of antigens on uveal melanomas differed markedly from primary cutaneous melanomas. The clinical relevance of these findings awaits further study.

Classification of routinely processed anaplastic large cell tumours with a small panel of antibodies. An immunohistochemical study with clinical follow-up.

A proportion of anaplastic large cell tumours is difficult to classify on sections of routinely processed, paraffin-embedded tissue. Differentiation into large cell lymphoma, carcinoma, melanoma or sarcoma is important in order to assess prognosis and proper treatment. Although the use of immunohistochemistry has been reported in the differentiation between some of these types of neoplasms, no antibody panel, which can directly differentiate all of them, has been described. In the present study we evaluated the value of a panel of 5 antibodies for the classification of 29 anaplastic large cell tumours, which could not be classified by experienced pathologists using conventional histological and histochemical techniques. The panel, which can be used on routinely fixed paraffin-embedded tissue, consisted of 5 different antibodies directed against keratin, vimentin, the human milk-fat globule membrane antigen MAM-6, a melanoma associated antigen and common leucocyte antigen. The use of this panel directly resulted in a definite diagnosis in 95% of the cases and provided valuable information for the diagnosis in the remaining cases. The diagnosis was confirmed by additional marker studies and electron microscopy. Moreover, clinical follow-up, including treatment data, was in accordance with the diagnosis based on the panel.

Correlation of histopathological characteristics with staining patterns in human melanoma assessed by (monoclonal) antibodies reactive on paraffin sections.

In order to investigate their possible role as prognostic markers, staining with the antibodies NKI/C-3 and anti-S100, that are applicable on paraffin sections, was examined using a group of primary cutaneous melanomas and autologous metastases using the immunoperoxidase procedure. All melanoma lesions stained with anti-S100, and the large majority with NKI/C-3. In primary melanomas showing a moderate or dense associated lymphocytic infiltrate, significantly more tumour cells stained with anti-S100 than in primary melanomas with a slight or absent infiltrate. In markedly pigmented metastases, significantly more tumour cells stained with NKI/C-3 than in less pigmented lesions; in primary melanomas this phenomenon just failed to be significant. In metastases with a high mitotic index a significantly lower proportion of tumour cells stained with NKI/C-3 than in lesions with a low mitotic index. No significant differences in staining were found between a group of primary melanomas with metastases and a group without metastases within a follow-up period of 5 years. Therefore, although staining with NKI/C-3 and anti-S100 appears to be associated with certain histopathological characteristics, it has no direct contribution to the assessment of prognosis in primary melanoma.

Circulating melanoma-associated antigen detected by monoclonal antibody NKI/C-3.

NKI/C-3 and NKI/black-13 are monoclonal antibodies recognizing different epitopes on a melanoma-associated antigen that is preserved after fixation in formalin and embedding in paraffin in virtually all melanoma tissues. The antigen, a predominantly cytoplasmic vesicle membrane-bound heterogeneous glycoprotein of 25-110 X 10(3) daltons, was shown to be a single 25 X 10(3) dalton polypeptide when incorporation of N-linked carbohydrates was inhibited by tunicamycin. The antigen was measured in a double determinant enzyme immunoassay (DDEIA) using NKI/C-3 as catcher antibody. Results from in vitro experiments indicated that the antigen is actively shed from living cells. In sera from melanoma patients with a small tumor burden, the antigen concentrations were in the range of those of controls (0-22 U/ml). Significantly increased values (33-600 U/ml) were found in sera from patients with a moderate or large tumor burden. The antigen concentrations in sera from patients with multiple metastases of other tumors were within the range of controls. Several sera from patients with multiple metastases of colon, pancreatic, and stomach carcinoma, however, contained increased antigen concentrations (45-80 U/ml). These results correspond with the reactions of NKI/C-3 in tissue sections of some malignancies other than melanoma. During the follow-up of melanoma patients the concentrations of circulating antigen correlated with tumor progression. The predictive value of the NKI/C-3 assay was no better than determination of serum lactate dehydrogenase, alkaline phosphatase or gamma glutamyl transferase activity.

Biochemical characterization and cellular localization of a formalin-resistant melanoma-associated antigen reacting with monoclonal antibody NKI/C-3.

A monoclonal antibody (MAb NKI/C-3) produced against a purified membrane preparation of human melanoma cells reacts preferentially with sections of formaldehyde-fixed and paraffin-embedded tissues of melanoma, nevocellular nevi, carcinoids and medullary carcinomas of the thyroid. NKI/C-3 did not react with basal-cell carcinoma, brain tissue or brain tumors, and in only 14/196 other tumors was a clear cross-reactivity observed, e.g. with prostate carcinomas and a minority of primary breast, ovarian, lung and clear-cell carcinomas. This antibody was used in an immuno-electron microscopic study for the cellular localization of the antigen. The antigen was dispersed in the cytoplasm of melanoma cells, and more concentrated inside vacuoles and sometimes also on the melanosomes. Occasionally, the antigen was seen on the cell surface. The nature of the antigen was determined in an enzyme immunoassay (EIA). It was found that the antigen is a glycoprotein with a disulfide-dependent configuration that is essential for recognition by the MAb. The antigen was distributed heterogeneously during gel filtration as well as during SDS-polyacrylamide gel electrophoresis in the region of 25-110 kd proteins. A purified antigen preparation that was obtained after affinity chromatography on a column of MAb NKI/C-3 linked to Sepharose 4B contained a carbohydrate:protein ratio of 1:3.5.

Immunohistochemical and histochemical tools in the diagnosis of amelanotic melanoma.

The histologic diagnosis of (metastatic) oligomelanotic or amelanotic melanoma may be difficult. In most cases this diagnosis can be established with conventional light and electron microscopic examination, supplemented with staining for melanin on ultrathin sections, but in other cases it remains equivocal. Therefore, the melanoma-associated monoclonal antibody NKI/C-3, effective on paraffin sections, was tested with an indirect immunoperoxidase technique. All 19 metastatic melanomas, used as positive controls, were stained. Seventeen of 23 primary melanomas and 8 of 9 initially equivocal eventually unequivocal melanomas (Group I) were stained with a diffuse cytoplasmic and in some cases locally peripheral pattern. Only two large cell undifferentiated carcinomas of 58 histogenetically unrelated but differential diagnostically relevant tumors showed localized staining in few tumor cells. Furthermore, 10 of 20 histogenetically related tumors (neuroendocrine tumors and clear cell sarcomas) were positive. These tumors however, can easily be differentiated from melanomas by other means. Of 15 equivocal melanomas (Group II) 9 cases reacted with NKI/C-3, suggesting that it may be a useful marker for difficult metastatic tumors suspect for amelanotic melanoma. Although sensitivity of NKI/C-3 for metastatic melanomas is high, its specificity is not sufficient. It therefore can be applied most properly in a selected panel of different tumor-associated antibodies that are reactive in formaldehyde fixed, paraffin-embedded tissue.

Antibodies and antigen expression in human melanoma detected by the immune adherence test.

By means of a modified immune adherence (IA) technique, sera from melanoma patients were tested for the presence of antimelanoma antibodies. In total 13/73 sera tested showed a positive IA reaction of which 4/6 sera showed a positive reaction in the autologous situation. Sera from 33 patients with other tumors, 7 patients with non-neoplastic diseases and 50 healthy individuals did not show any IA reactivity towards melanoma cells. The reaction seemed to be selectively directed against tumor-associated antigens (TAA) on melanoma cells. No correlation with the stage of the disease could be found. Longitudinal studies indicated that conversions in antibody activity did not correlate with the clinical state of the patients. There was also no correlation with the corresponding in vitro data obtained in cell-mediated immunity tests. Cell lines and short-term cultures originating from tumors from different melanoma patients shared a common antigenicity. The expression of TAA on cells from a melanoma cell line fluctuated significantly during prolonged culture. The expression of TAA was influenced by the culture conditions and the growth state of the cells. A relation between TAA-expression and cell cycle phase was demonstrated.